A systematic exploration reveals the potential of spermidine for hypopigmentation treatment through the stabilization of melanogenesis-associated proteins

Spermidine (SPD), a polyamine naturally present in living organisms, is known to prolong the lifespan of animals. In this study, the role of SPD in melanogenesis was investigated, showing potential as a pigmenting agent. SPD treatment increased melanin production in melanocytes in a dose dependent manner. Computational analysis with RNA-sequencing data revealed the alteration of protein degradation by SPD treatment without changes in the expressions of melanogenesis-related genes. Indeed, SPD treatment significantly increased the stabilities of tyrosinase-related protein (TRP)-1 and -2 while inhibiting ubiquitination, which was confirmed by treatment of proteasome inhibitor MG132. Inhibition of protein synthesis by cycloheximide (CHX) showed that SPD treatment increased the resistance of TRP-1 and TRP-2 to protein degradation. To identify the proteins involved in SPD transportation in melanocytes, the expression of several solute carrier (SLC) membrane transporters was assessed and, among 27 transporter genes, SLC3A2, SLC7A1, SLC18B1, and SLC22A18 were highly expressed, implying they are putative SPD transporters in melanocytes. Furthermore, SLC7A1 and SLC22A18 were downregulated by SPD treatment, indicating their active involvement in polyamine homeostasis. Finally, we applied SPD to a human skin equivalent and observed elevated melanin production. Our results identify SPD as a potential natural product to alleviate hypopigmentation.


Results
SPD treatment increases melanin production. SPD is a polyamine compound (C 7 H 19 N 3 ) that exerts various metabolic functions in living organisms (Fig. 1a). To investigate the effect of SPD on melanogenesis, normal human primary melanocytes and a human melanoma MNT-1 cell line, with the capacity to produce all stages of melanosomes, were obtained. The cytotoxic effects of SPD were evaluated by treating MNT-1 cells with various concentrations of SPD (Fig. 1b). It was confirmed that cell viability was affected only at concentrations of 50 and 100 μM. Therefore, in this study, SPD was administered in concentrations below these values. Quantification of melanin content in MNT-1 cells treated with increasing concentrations of SPD revealed increased melanin production in these cells (Fig. 1c). Moreover, based on a previous report indicating that SPD exerts distinct effects in young vs. aged tissues 31 , young and aged primary human melanocytes were treated with SPD. SPD increased melanin production in both cell types (increments of 15 ± 5% and 12 ± 2% in young and aged cells, respectively) (Fig. 1d,e).

SPD treatment modulates genes involved protein degradation.
To further understand the molecular implications of SPD treatment, the effects of this polyamine were analyzed through a systematic approach. Firstly, RNA sequencing performed in SPD-treated cells revealed the modulation of only 181 downregulated and 82 upregulated genes (Fig. 2a), of which melanogenesis-related genes were not included. To further confirm this, we next analyzed the effects of SPD on the expression levels of tyrosinase family genes TYR , TRP-1 and TRP-2, which tightly regulate melanogenesis (Fig. 2b). mRNA expression levels confirmed that this polyamine did not alter melanogenesis-associated gene expression. However, we found that several of the genes whose activity was altered by SPD were related to protein degradation (Fig. 2c). Several of the altered genes are involved in ubiquitination, a system for degradation of proteins that is also associated with the degradation of melanogenesis www.nature.com/scientificreports/ proteins. Altogether, these data infer that SPD does not increase melanogenesis by upregulating its associated genes' expression levels, but suggests it might influence melanogenesis by modulating protein stability.
SPD treatment improves the stabilities of TRP-1 and TRP-2. Since melanin production is regulated by a balance of synthesis and degradation of melanogenesis-associated proteins, we next aimed to better understand the effects of SPD at the protein level. SPD treatment resulted in a significant increase in TRP-1 and TRP-2 protein levels but not TYR (Fig. 3a). Moreover, since the proteasome system is involved in the degradation of melanogenesis-related proteins 32 , the expression of ubiquitin under SPD treatment was analyzed and revealed an altered ubiquitin expression pattern. This result, which is consistent with the RNA-seq data (Fig. 2c), may indicate that SPD increases melanogenesis by decreasing protein degradation via ubiquitination. Moreover, after treatment with SPD and subsequent application of the proteasome inhibitor MG132, expression levels of  www.nature.com/scientificreports/ TRP-1 and TRP-2 were also significantly increased, unlike TYR, and ubiquitin expression decreased. These data further demonstrate that SPD modulates ubiquitination in addition to confirming that it has no impact on TYR expression. In addition, confocal microscopy revealed accumulation of the late-stage melanosome marker TA99 in cells treated with SPD ( Fig. 3b), indicating the accumulation of highly pigmented melanosomes in these cells. Finally, to block the transcription of melanogenesis-related genes, cycloheximide (CHX) was applied (Fig. 3c). Under these conditions, SPD treatment improved the stabilities of TRP-1 and TRP-2, further demonstrating  www.nature.com/scientificreports/ that SPD increases melanogenesis by improving protein stability which, in turn, increases TRP-1 and TRP-2 expression levels.
Identification of putative SPD transporters in primary human melanocytes. Intracellular polyamine levels are regulated by a combination of their synthesis, catabolism, and transport 33 . In order to identify the proteins involved in SPD transportation in melanocytes, the expression of several solute carrier (SLC) membrane transporters was assessed since they are reportedly involved in polyamine transportation 12,[34][35][36][37][38] . Firstly, transcriptome data ( Fig. 4a) revealed that, among the 27 transporter genes, SLC3A2, SLC7A1, SLC18B1, and SLC22A18 were highly expressed in primary human melanocytes. Since the balance of a substrate is maintained due to the selective action of transporters, and transporter protein expression levels decrease under high substrate concentration as a strategy to maintain ideal cytosolic levels and avoid cytotoxic effects 39 , we next aimed to observe the mRNA expression levels of these three genes under SPD treatment (Fig. 4b). Interestingly, SLC7A1 and SLC22A18 were the only genes responsive to the treatment, indicating their active involvement in polyamine homeostasis. Hence, SPD supplementation leads to an increase of the cellular polyamine pool, mediated by the actions of SLC3A2, SLC7A1, SLC18B1, and SLC22A18, thus increasing the stability of TRP-1 and -2 (Fig. 4c). These data demonstrate the potential role these transporters play in polyamine homeostasis to support protein stability for melanogenesis.

SPD increases melanogenesis in vivo.
Finally, we aimed to analyze the potential of applying SPD as a treatment for hypopigmentation. We confirmed the effects of SPD in vivo by applying this polyamine to a human skin equivalent containing melanocytes. SPD supplementation resulted in a significant increase in melanin levels (increment of 10 ± 5%), confirming its potential application for treatment of hypopigmentation disorders (Fig. 5).

Discussion
In the present study, a systematic exploration approach revealed that SPD treatment modulates genes involved in protein degradation. Furthermore, protein analysis demonstrated that the melanogenesis-related proteins TRP-1 and TRP-2 were stabilized following SPD treatment. Finally, a human skin equivalent model revealed that SPD increased melanin production in vivo. Therefore, our findings demonstrate the potential of SPD for ameliorating hypopigmentation. The polyamine pool gradually declines with age 33 . A previous study analyzed polyamine levels in 14 different tissues of an aging mouse model and revealed that SPD levels dropped in 11 of the 14 tissues 40 . SPM decreased only in the skin, heart, and muscles, while PUT levels were remarkably low in all tissues and at all ages. Indeed, SPD plays important roles in many tissues, and SPD supplementation leads to the relief of several age-related conditions. Moreover, hypopigmented macules, such as stellate pseudoscars and idiopathic guttate hypomelanosis, are frequently observed in photodamaged skin of aged individuals, and hair graying is a common feature of aging 41 . With this in consideration, since aged cells have a deficient polyamine pool, it seems reasonable that SPD treatment can restore it to more optimal levels thus increasing melanogenesis. However, our results also demonstrate that SPD treatment led to melanogenesis improvement in young cells. The beneficial effects of SPD supplementation in young mouse models have also been previously reported 42 . Thus, this infers that the benefits of SPD treatment are not limited to aged individuals, despite the polyamine pool knowingly decreasing during aging, and may also target non-age-related hypopigmentation, or premature hair graying. Hence, we state that SPD is a promising treatment to alleviate hypopigmentation, in which therapy doses should be customized to each patient for maximum efficacy.
Melanogenesis in human skin is tightly regulated by the expression of melanin-associated genes and the subsequent enzymatic reactions caused by these genes. Our results revealed that SPD treatment improved melanogenesis without altering the expressions of melanogenesis genes. Instead, SPD altered the expression of genes related with protein degradation, including proteasome-associated genes. Simultaneously, we observed an improvement of the stability of the melanogenesis proteins TRP-1 and -2 and decreased ubiquitination after SPD application. Indeed, melanogenesis proteins are digested by the proteasome system 27,43,44 . Ubiquitination is a system that targets eukaryotic proteins for breakdown and is dependent on a cascade of E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes and E3 ubiquitin-protein ligases that subsequently bind the target protein for degradation in the proteasome 15 . SPD has been implied in the impairment of ubiquitination by inhibition of ubiquitin ligases 45 , thus this is a plausible explanation for why SPD stabilizes TRP-1 and TRP-2. Interestingly, SPD did not significantly affect TYR expression. A previous study that observed the degradation rates of TYR, TRP-1, and TRP-2 revealed that TYR has a greater half-life than TRP-1 and TRP-2 46 . Therefore, TYR may have a more rigid protein structure that confers it better stability. Another factor to take into consideration is that protein stability is also influenced by differences in the degradation machinery. Humans are estimated to possess 500-1000 E3 ubiquitin ligases, which are thought to play a key role in protein identification 47 . In this regard, it is also possible that TYR might be tagged by different ubiquitin ligases from those binding TRP-1 and TRP-2. Further research about the distinct structures of these proteins and their ubiquitin binding sites is necessary to answer these questions. Furthermore, fatty acids have been shown to regulate melanogenesis 48 . Similar to our findings, palmitic acid treatment of melanocytic cells led to decrease in ubiquitination, which increased the stability of melanogenesis proteins without alterations of their mRNA levels. However, in contrast with our work, palmitic acid treatment increased TYR expression, but not TRP-1 and -2. It is interesting to note that one of the molecular targets of SPD is lipid metabolism, which is regulated through autophagy 49 . Indeed, activation of autophagy has been linked with induction of melanogenesis and regulation of melanosome biogenesis in melanocytes 50 . Therefore, we cannot rule out the possibility that SPD might also alter the stability of melanogenesis proteins through www.nature.com/scientificreports/ modulation of autophagy. However, since our bioinformatics data did not identify changes in autophagy after SPD supplementation, we did not focus on that mechanism in this investigation. This may be due, in part, to the fact that we used a lower dose of SPD than other researches, which was based on the results of the cell viability assay. The modification of lipid metabolism following SPD treatment needs to be further investigated in order to fully comprehend these events. Nonetheless, our results infer that targeting protein stability could constitute a novel therapeutic strategy to overcome hypopigmentation. www.nature.com/scientificreports/ Since polyamines are positively charged at physiological pH, they require a transport system to take up exogenous polyamines and/or eliminate excess polyamines from the cell 51,52 . Moreover, while the mechanisms involved in polyamine biosynthesis and catabolism have been thoroughly studied, the mammalian polyamine transport system is still poorly known. The SLC superfamily, which has 384 members, is the second-largest group of membrane transporter proteins in the human genome 53 . SLC transporters are primarily involved in the transportation of small molecules into cells by moving substrates via ion or electrochemical gradients like sodium or proton gradients 54 . Several SLC transporters have been implied in polyamine transportation 36 . Despite that, the SPD transporters in melanocytes had not been defined yet. We observed that SLC3A2, SLC7A1, SLC18B1, and SLC22A18 transporters genes were highly expressed in melanocytes. Moreover, since substrate balance is maintained by the selective action of transporters, we hypothesized that SPD supplementation would downregulate their expression levels to maintain cytosolic levels. However, only SLC7A1 and SLC22A18 were responsive to SPD treatment. As a result, we can imply that these two proteins may be actively involved in SPD-dependent polyamine homeostasis in melanocytes. In future works, a more thorough analysis will be required to better define the SPD transporters in melanocytes. Cell-based transport assays are limited since various endogenous transporters can disturb the transportation of a target molecule. Instead, proteoliposome-based transport assay performed with a purified pure transporter offers an effective model system for investigating the transportation of a target molecule 55 . Prior to reconstructing a proteoliposome, expression and purification investigations should be addressed. These methodologies present the potential to complement our investigation and precisely identify the SPD transporters in melanocytes.
In conclusion, this study indicates that SPD is a promising compound for the treatment of hypopigmentation disorders through improved protein stability, thus demonstrating great economical value given the current market demand for natural and human-friendly cosmetic products.

Melanin assay.
Melanocytes were seeded at a density of 1 × 10 5 cells/dish. After overnight incubation, cells were treated with SPD for 7 days. Cells were lysed in 0.5 M Tris-HCl (pH 7.5) containing 1% NP-40, 0.15 M NaCl, and 5 mM MgCl 2 , as previously described 56 . Cell lysates were centrifuged at 15,000 rpm and 4 °C for www.nature.com/scientificreports/ 20 min. The protein content of each supernatant was measured using a bicinchoninic acid (BCA) assay, and 100 μL of NaOH solution was added to dissolve each pellet and heated at 80 °C for 1 h. Melanin levels in the cells were determined by measuring absorbance at 450 nm. Absorbances were divided by the amount of protein used to determine each total melanin content.
Western blotting analysis. Cells

Identification of differentially expressed genes (DEGs).
To identify DEGs, a statistical hypothesis test was performed 57 . For each gene, a T-statistic value was calculated using the Student's t-test for comparison of saline-and SPD-treated melanocytes. An empirical distribution of the T-statistic value for null hypothesis was estimated by performing all possible combinations of random permutations of the samples. Adjusted P-values were obtained for each gene using a two-tailed Student's t-test with an empirical null distribution. The DEGs were identified as genes with adjusted P-values ≤ 0.05, and absolute log 2 -fold changes > 0.428 (1.35-fold; the mean of the 0.5th and 99.5th percentiles of the null distribution of log2-fold changes).

Human skin equivalent.
A human skin equivalent model composed of primary human keratinocytes and melanocytes (KeraSkin™, Biosolution, Korea) was used. The basal layer of this human skin equivalent model is composed of dendritic cells that produce melanin granules. These cells produced pigmentation and became fully differentiated when cultured in an air-liquid interface culture for 2 weeks. Cells were then treated with 2 and 4 µM SPD for 2 weeks.
Statistical analysis. Two-tailed Student's t-test was used to analyze the differences between two groups.

Data availability
The raw and processed RNA-seq data were deposited into the Gene Expression Omnibus (GEO) database with accession ID GSE209538.